The goal of this proposal is to understand the membrane-proximal events that lead to lytic function in Natural Killer (NK) cells. NK cells are key players in innate immunity and are responsible for early defense against intracellular bacteria, viruses and tumors. Despite major developments in the identification of NK receptors and their ligands, the signal mechanisms triggered by these interactions have not been fully resolved. NK receptors have no intrinsic kinase activity and rely on adaptors to initiate the signal process leading to lytic function. The adaptor itself has no kinase function but, in the case of DAP12, zeta, FcepsilonRgamma, it bears an immunoreceptor tyrosine-based activatory motif (ITAM), which allows for binding to Syk/Zap70.On the other hand, DAP10 has a binding site for PI 3-kinase. It is unknown how DAP 10 and DAP12 become activated. Based on preliminary data focused on DAP 12, target cell engagement activates Fyn in NK cells and dominant-negative (DN) Fyn but not DN-Lck blocks lyric function in NK cells. In addition, Syk but not Zap70 is activated in this system. However, in another NK system that utilizes DAP10, we have suggestive data that Lck can also be involved. We propose the hypothesis that a specific Src kinase present in NK cells (i.e. Lck, Fyn, or Lyn) and Syk or Zap70 can be selected, depending on the NK receptor/adaptor combination. Aim 1 defines the mechanism of DAP12 activation and its control by different NK receptors. The binding site for Src kinases on DAP12 or the NK receptor, and the identification of the tyrosines on DAP12 subject to Src kinase phosphorylation will be analyzed using immunoprecipitation/western blotting systems. GST fusion proteins, deletion mutants, and site-directed mutagenesis will be employed. Aim 2 analyzes the molecular basis for Syk/Zap70 phosphorylation. Co-expression of Src kinase mutant constructs, DAP12, Zap 70, and Syk in 293 cells will help to identify if the identified Src kinase directly phosphorylates Syk/Zap70 or requires DAP 12 as an adaptor to recruit Syk/Zap70 for subsequent phosphorylation. Aim 3 analyzes the mechanisms of DAP 10 activation and of the dual ability of NKG2D-S to bind DAP10 and DAP12. Aim 4addresses the involvement of lipid rafts and cytolytic synapes in propagating the lytic signal cascade. Aim 5 analyzes lytic signal pathways in NK cells of LGL leukemic patients to determine if the signal pathways uncovered for each of the Nk receptor/adaptor pair are universally applicable. Through these studies, important insight will be gained into how the various classes of NK Receptors provide signal specificity that regulates lytic function.